This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The clam Scapharca inaequivalvis possesses two hemoglobins that represent exceptional model systems for the investigation of protein allostery. Both hemoglobins bind oxygen cooperatively using a structural mechanism that is very different from the more well studied human hemoglobin. The dimeric hemoglobin termed HbI is the simplest possible model system for allostery with two identical subunits. Time-resolved crystallographic analysis of this hemoglobin provided for the first time a preliminary structural description of allosteric changes in real time (Knapp et. al. 2006 PNAS 103 7649-7654). Despite the overall success of these experiments a major drawback was the very high level of geminate rebinding in the crystal which substantially reduced the signal during the allosteric transition. Our analysis of ligand migration including time-resolved crystallographic experiments solution experiments and computational analysis (Knapp et al. 2009 Structure 17 in press) strongly suggests the crystal lattice restricts ligand exit by damping transient subunit rotations that are required for exit through a distal histidine gate. These experiments also revealed a potential alternate exit route through a "back door" channel. We are producing mutants that will allow ligands to exit through this back door within the tight confines of the crystal lattice. One of these has already been shown by optical experiments to reduce geminate rebinding in crystals. We propose to use such mutants with the substantially upgraded BioCARS beamline 14-IDB to obtain significantly improved understanding of the progression of structural events that underlie cooperative oxygen binding. This will for instance allow us to define leading and lagging components of the structural transitions to identify those structural events that trigger later events. Moreover we intend to use allosteric mutants to elucidate protein relaxation in alternate T and R states and to dissect individual structural components of the allosteric transition. The tetrameric hemoglobin termed HbII is formed from two heterodimers each of which has a similar assembly to that of HbI. The presence of two different subunits will permit investigation of how one subunit impacts a second subunit which is not possible in the two-fold symmetric HbI. Therefore we propose to use time-resolved x-ray diffraction experiments to elucidate the kinetic structural pathway in the tetrameric HbII and specific mutants of HbII. Mutants will allow us to separate out the effects of one subunit type on the second subunit either by altering the geminate recombination properties or by locking one subunit in a high affinity or low affinity state. Like HbI but unlike human hemoglobin we have recently shown that Scapharca HbII crystals can undergo the full allosteric transition within crystals. As a result this system is well suited for time-resolved crystallographic experiments of allosteric protein function. Allosteric transition will be triggered by laser photolysis of CO-liganded hemoglobin crystals. At various time points ranging from 100 picoseconds to 100 microseconds diffraction data will be collected by Laue methods. The structures obtained at these time points will reveal the kinetic pathways as the protein undergoes its allosteric transition from the liganded to the unliganded form.